Ex Vivo method for determination of CETP activity and efficacy of heart disease treatment

ABSTRACT

The present invention relates to a novel method to determine CETP activity in a bodily fluid sample wherein the sample may contain an inhibitor of CETP activity. The effect of the inhibitor is preserved to provide a more accurate determination of CETP activity in the fluid. Kits for the practice of the method are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) toprovisional application 60/372,628, filed Apr. 11, 2002, which is herebyincorporated by reference as if fully set forth.

TECHNICAL FIELD

[0002] This invention relates generally to methods of determiningCholesteryl Enzyme Transfer Protein (CETP) activity in bodily fluids ofa subject with increased accuracy by reducing undesirable dilutioneffects present in other assays for CETP activity. The methods areapplied in the form of heart disease test kits and methods ofdetermining heart disease risk factors as well as methods of determiningthe efficacy of treatments for heart disease. The present invention alsorelates to a method and kit for determining an atherosclerosis riskfactor as well as methods and kits for determining the efficacy of atreatment regimen for abnormalities associated with CETP activity.

BACKGROUND ART

[0003] It is known that heart disease or atherosclerosis is a result ofthe build up and subsequent restriction of blood vessels by plaque ofcirculating blood to the heart muscle. It is believed a preemptivefactor to plaque development is depositing or loading of the cellscomprising the blood vessel lining with cholesterol in the form ofcholesteryl ester. It is also believed the cholesteryl ester originatesfrom sources that include lipoproteins, specifically the low density(LDL) and very low density (VLDL) lipoproteins. The high densitylipoproteins (HDL), however, are known to be protective elements whereheart disease or atherosclerosis is concerned and are desirablelipoproteins.

[0004] The medical profession utilizes several methods to minimize theexposure of cells that line the circulatory system to VLDL and LDL withthe rationale that lowering the exposure of arterial walls to LDL andVLDL will lower the ability of the cells to load or uptake cholesterylester from these lipoproteins. Means of lowering LDL and VLDL includeindirect means such as disruption of the cholesterol biosyntheticpathway that occurs within the liver cells. Drugs, such as the statins,including Mevacor® and Lipitor® inhibit HMG-Coenzyme A reductase, a keyenzyme along the cellular pathway for production of cholesterol.

[0005] Cholesterol is a life sustaining component necessary to maintainmany basic functions such as cell membrane integrity. Cholesterol istransported throughout the circulatory system within lipoproteins in theester form as cholesteryl ester. The ester is completely water insolubleand resides in the core of the lipoproteins. Statin drugs are given inlimited doses so that the pathway is not totally without output ofcholesterol but the output is partially reduced. The reduced cholesteroloutput from the liver results in generally a reduction of allcholesterol pools or stores. Included is the reduction of thelipoprotein cholesterol pool. Lipid transfer proteins or other proteinsthat utilize lipids as substrates interact with the lipoproteins toaccess their substrates both at the core and the surface of thelipoprotein. One lipid transfer protein, in particular a neutral lipidtransfer protein, cholesteryl ester transfer protein (CETP), whichshuttles cholesteryl esters from HDL to VLDL and LDL.

[0006] There are various known techniques to measure cholesteryl estertransfer protein (CETP) activity. For example, an article entitled:Effect of Very Low-Density Lipoproteins on Lipid Transfer in IncubatedSerum, by A. V. Nichols and L. Smith, J. Lipid Research, vol. 6, pp.206-210 (1965), measures the activity of CETP by determination ofcholesteryl ester (CE) mass transfer. The determination of CE masstransfer from high density lipoprotein to very-low density lipoprotein(VLDL) and low density lipoprotein (LDL) requires the re-isolation ofVLDL and LDL after incubation with HDL and the CETP source in order todetermine the cholesteryl ester mass transfer.

[0007] The VLDL/LDL re-isolation from the incubation mixture is atechnique that includes ultra-centrifugation of the incubation mixturefor many hours so that the VLDL and LDL components are floated upwardsthrough a density gradient as the HDL component of the incubationmixture sinks to the bottom of the centrifuge tube. Further processingof the sample requires a method of determining the amount or mass ofcholesteryl ester associated with the re-isolated VLDL or LDL andequating a change in mass to CETP facilitated transfer. Later variationsof this method of activity measurement have simplified massdetermination by utilizing HDL that has a radioactive label associatedwith the CE.

[0008] An article entitled: Cholesteryl Ester Exchange Protein in HumanPlasma Isolation and Characterization by N. M. Pattnaik, A. Montes, L.B. Hughes and D. B. Zilversmit, Biochemica et Biophysica Acta 530, pp.428-438 (1978), discloses a method of activity measurement of CETP thatalso utilizes radioactive CE in HDL. This method is an improvement overthe above method by simplifying the incubation mixture componentsseparation or the re-isolation technique discussed above. In the citedarticle, separation of the LDL component from the HDL component isaccomplished by precipitation of the LDL component of the incubationmixture. The LDL precipitate is pelleted by a relatively shortslow-speed centrifugation and the remaining HDL supernatant is counted.The loss of radioactivity from the HDL component is attributed to 3H-CEtransferred to the LDL pellet.

[0009] An article titled: Fluorescent Determination of Cholesteryl EsterTransfer Protein (CETP) Activity in Plasma by N. Dousset, L.Douste-Blazy in Clinical Chemistry, vol. 38, No. 2, p. 306 (1982), is animprovement over previous methods of activity measurement since itdiscloses a technique that does not require radioactive components. Inthis method, transfer activity of the CETP is determined by themeasurement of transfer of a fluorescent labeled CE. In this reference,the cholesteryl ester molecule utilized as the CETP substrate fortransfer has been covalently bound to a fluorescent molecule derivedfrom pyrene. The pyrene labeled cholesteryl ester (PY-CE) is recognizedby the CETP and the PY-CE may be detected by a fluorimeter. Theaccumulation of the PY-CE in the LDL fraction is, however, only able tobe determined after the separation of the LDL acceptor from the HDLdonor.

[0010] An article entitled: Enhancement of The Human Plasma LipidTransfer Protein Reaction by Apolipoproteins by T. G. Milner, K. W. S.Ko, T. Ohnishi, and S. Yokoyama in Biochimica Biophysica Acta 1082, pp.71-78 (1991), discloses a method for determining the activity of CETPalso utilizing a pyrene labeled CE (PY-CE). This method does not requireseparation or re-isolation of substrates, but uses the measurement ofboth monomer and excimer fluorescent emission from the pyrene label todetermine a ratio thereof. The cited article improves upon certainaspects of the previous method.

[0011] An article entitled: Use of Fluorescent Cholesteryl EsterMicroemulsions in Cholesteryl Ester Transfer Protein Assays by CharlesL. Bisgaier, Laura Minton, Arnold D. Essenberg, Andrew White, andReynold Homen published in the Journal of Lipid Research, Volume 34,1993 discloses a method that utilizes a self-quenching fluorescentlabeled cholesteryl ester core.

[0012] Additionally, there are methods disclosed by the present inventorin U.S. Pat. Nos. 5,770,355; 5,618,683; 5,585,235 and 6,174,693.

[0013] Citation of the above documents is not intended as an admissionthat any of the foregoing is pertinent prior art. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicant and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

DISCLOSURE OF THE INVENTION

[0014] The invention is directed to assays for CETP activity in bodilyfluids of a subject, which methods have improved accuracy. The inventionis based in part on the recognition that inhibitors of CETP activity maybe present in bodily fluids and that dilution of the bodily fluids in anassay method dilutes such inhibitors to result in an inaccurateassessment of CETP activity in the bodily fluid. Inhibitors of CETP maybe present in the bodily fluids of a subject as a result of variousfactors, including, but not limited to, administration of a CETPinhibitor (or prodrug form thereof) to said subject, endogenousinhibitors that are ordinarily present in said subject, and endogenousinhibitors that are induced by administration of a medication, dietaryfactors, and/or physical activity. There are reports of endogenousinhibitors of CETP (see for example, U.S. Pat. No. 5,512,548) inaddition to chemical inhibitors (see for example, U.S. Pat. No.6,313,142). Such inhibitors may include apoC-I.

[0015] Previous methods fail to allow the measurement of CETP activityin a bodily fluid, such as plasma, when an inhibitor is also present inthe fluid at an effective concentration. This failure is because thosemethods require dilution of the plasma or serum sample in the assayused. This results in the dilution of the inhibitor below theconcentration that was originally present in vivo and thus possiblybelow the concentration where it inhibits CETP. This dilution effectcreates a problem in obtaining an accurate determination of CETPactivity in the bodily fluid because it is not readily possible todetermine the identities, and amounts thereof, of inhibitors present ina sample of bodily fluid from a subject, such as a human patient. Thesame problem arises whenever a sample of bodily fluid is added to anassay of a volume significantly greater than the sample volume.

[0016] The present invention provides an ex vivo assay that does notrequire significantly alterations in the concentration of a CETPinhibitor that may be present in a bodily fluid sample. Thus moreaccurate measurements of CETP activity in a bodily fluid sample may bemade despite the presence of an inhibitory drug or compound in thesample. Of course the invention may also be practice with samples thatare free of exogenously supplied CETP inhibitors to provide a moreaccurate determination of CETP activity in a bodily fluid.

[0017] Thus in one aspect, the present invention provides a method forthe determination of CETP activity in a bodily fluid sample, such as aplasma sample, without dilution of the sample in order to make CETPactivity measurements in samples that may or may not be treated withCETP inhibitors. In addition to preventing dilution of possibleinhibitors of CETP activity, the present invention permits measurementof CETP activity in biological fluids without significant alteration ofthe concentration of the components of the biological fluid.

[0018] In one embodiment, the ex vivo method includes 50 microliters ofa bodily fluid, such as a plasma sample, to be assayed and 4 microlitersof assay reagents in a homogeneous CETP activity assay. Of course largerand smaller volumes, such as from about 10 to about 100 microliters, ofa bodily fluid may be used in the present invention, which providesassay conditions where the physiological sample is present as a majorityof the total assay volume. Preferably, the majority is about 90% orgreater of the assay volume. These conditions are preferably applied tomeasure the effect of a drug at a specific concentration in a patient'splasma sample and to collect data regarding the effect of the drug onCETP activity at specific concentrations. The assay reagents comprise adonor particle and an acceptor wherein the donor particle contains asubstrate for transfer by CETP. The substrate is preferably labeled suchthat the transfer of the substrate from the donor particle to anacceptor is detected as an indicator of CETP activity.

[0019] Given the role of CETP activity in disorders such as coronaryartery lesions and others involving cholesteryl ester deposits, thepresent invention is preferably applied to solve a variety of problemsfacing health care providers such as physicians, clinical hospitalstaff, laboratories, pharmaceutical companies and the like, in thediagnosis of risk factors for heart disease and atherosclerosis. Theinvention provides hundreds of millions of individuals who are at riskof developing heart disease and atherosclerosis, either as a result ofgenetic factors, diet, or the like, with a simple diagnostic tool andmethod for more accurately diagnosing risk factors for these conditions.The invention also provides means to determine the efficacy oftreatments of lipid transfer protein disorders and in particular CETPrelated disorders such as coronary artery lesions. The invention targetsthis group of hundreds of millions of individuals worldwide by providinga method and kit for determining a heart disease risk factor, namely,abnormal activity of CETP, and a method and kit for determining theefficacy of a treatment that targets CETP activity.

[0020] The present invention is preferably embodied as a non-radioactivemethod and kit to simplify, facilitate the determination of, andquantify a risk factor for heart disease, and in particularatherosclerosis and other disorders involving cholesteryl esterdeposits. The invention may also be used to determine the efficacy oftreatments affecting the activity of and abnormalities in lipid transferprotein activity in physiologic samples of CETP.

[0021] In another aspect, the present invention is applied as methods ofidentifying a subject as having a risk factor for heart disease,atherosclerosis, coronary artery lesions, and/or disorders involvingcholesteryl ester deposits by determining CETP activity in a bodilyfluid from said subject. These methods are optionally practiced withreference to predetermined standard values for CETP activity in bodilyfluids of subjects having these conditions. The invention also provideskits comprising components used in these methods in combination withinstructions for practicing the methods.

[0022] In a further aspect, the invention provides for the use of animproved donor particle emulsion for assessing CETP activity. Theimproved donor particle emulsion does not contain apoA-I, which is themajor protein component of high-density lipoprotein (HDL). CETPtransfers cholesteryl esters from HDL to LDL or VLDL (apoB-containinglipoproteins). The invention is thus also based in part on therecognition that donor particle emulsions that are apoA-I free do notcompete with endogenous HDL, which is present in a bodily fluid invarying amounts. The invention thus provides for reducing or eliminatingcompetition from endogenous HDL, which contributes to inaccuratedeterminations of CETP activity in assays that use apoA-I containingdonor particles. Such assays are hindered by the fact that the apoA-Icontaining donor particles will be of specific activities that varydepending on the amount of HDL present in a bodily fluid.

[0023] In one preferred aspect, the invention provides a method todetermine CETP activity in a bodily fluid of a subject, said methodcomprising obtaining a sample of bodily fluid from said subject,contacting said bodily fluid with a donor and an acceptor to form areaction mixture solution, wherein said bodily fluid comprises at leastabout 89% v/v of said reaction mixture solution, and detecting transferfrom said donor to said acceptor to determining the CETP activity insaid fluid. The donor and acceptor are preferably in solution togetherbut may also be in separate solutions that are combined with said bodilyfluid.

[0024] In another preferred aspect, the invention provides a method ofdetermining and quantifying CETP activity in a bodily fluid comprisingobtaining a sample of bodily fluid (preferably plasma) from a mammalhaving a source of CETP, incubating the sample, without significantdilution, in a non-radioactive CETP assay for an effective time periodto obtain an incubated mixture, measuring the CETP activity of theincubated mixture to determine a CETP activity value, and comparing thesample's CETP activity value to a predetermined standard value. Anon-radioactive CETP assay used in the practice of the inventionpreferably comprises using a prepared sonicated particle comprising afluorescently labeled cholesteryl ester and at least one apolipoproteinto a buffer to form a buffered solution, and an emulsion of lipid addedto the buffered solution to accept the transfer of labeled chloesterylester. After addition of the CETP containing sample to the bufferedsolution and passage of sufficient time, the fluoresence of the bufferedsolution can be measured and used as an indicator of CETP activity inthe sample.

[0025] In an embodiment of the above, 50 microliters of afrozen-then-thawed human plasma sample is combined with 4 microliters ofa solution containing a donor particle emulsion and acceptor asdescribed herein. The combination is incubated for about 45 to about 90minutes at 37° C. followed by detection of fluorescence at 535 nm withexcitation at 465 nm.

[0026] In a further preferred aspect, a non-radioactive method offacilitating, simplifying and quantifying a determination of theefficacy of a compound that modulates (activates or inhibits) CETPactivity, as well as a kit for carrying out the method, are provided.The method comprises obtaining a sample of bodily fluid (preferablyplasma) from a mammal having a source of CETP and having beenadministered a CETP modulating compound, incubating the sample, withoutsignificant dilution, in a non-radioactive CETP assay for an effectivetime period to obtain an incubated mixture, measuring the CETP enzymeactivity of the incubated mixture to determine a CETP activity valueand, comparing the sample's CETP activity value to a predeterminedstandard value. The method and kit are used to determine the efficacy ofa treatment designed to affect CETP enzyme activity. The treatmentincludes a compound that modulates the transcription of a gene codingfor CETP, a compound that modulates the translation of a gene coding forCETP, a compound that is a treatment for atherosclerosis, a modificationin diet affecting CETP activity, atherogenic diet modificationsaffecting CETP activity, or combinations thereof. Preferably, thecompounds are inhibitors of CETP activity or expression levels in bodilyfluids. Alternatively, the compounds are those used to treat heartdisease, such as the statin drugs (including Mevacor® and Lipitor® andothers that inhibit HMG-Coenzyme A reductase), that have beenadministered alone or in combination with a compound that affects CETPactivity.

[0027] In other aspects, the invention provides a non-radioactive kitand method of determining an HDL/LDL risk ratio or a risk factor forobesity as well as a kit and non-radioactive method to determine theefficacy of a treatment for a medical condition associated with abnormallevels of CETP. In all of these applications, the methods of theinvention are employed to obtain a value representing CETP activity in abodily fluid sample, which value is compared to a pre-determined range.The pre-determined range may include regions of low CETP activity,moderate CETP activity, and high CETP activity which have beencorrelated with HDL/LDL risk ratios and/or a risk factor for obesity.Alternatively, the pre-determined range is correlated with medicalconditions associated with abnormal levels of CETP, including, but notlimited to, hypoalphalipoproteinemia, abnormal and normal ratios ofplasma apo A-I to apo B.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 is a cross section of one donor particle emulsion accordingto the invention.

[0029]FIG. 2 is a cross section of a preferred donor particle emulsionaccording to the invention.

[0030]FIG. 3 is a cut away view of an acceptor lipoprotein used in theinvention.

[0031]FIG. 4 is a schematic illustration of the CETP transfer activityassay of the invention.

[0032]FIG. 5 is a graph of CETP activity in plasma as affected bypre-treatment with a CETP inhibitory monoclonal antibody.

MODES OF CARRYING OUT THE INVENTION

[0033] The invention provides the ability to more accurately determineCETP activity in samples of bodily fluids from a subject. Such bodilyfluid samples may be any that contains CETP activity and thus can serveas a source of CETP activity. Non-limiting examples of such bodily fluidsamples include any biological fluid obtained or derived from a subject,whole blood, plasma, serum and extracts, homogenates, or secretions ofliver, small intestine, spleen, adrenal gland, adipose tissue, as wellas combinations of such extracts, homogenates, or secretions.Preferably, the subject is human, although other vertebrates,particularly mammals, under veterinary care may be the subjects of thepresent invention. Particularly preferred are animals important to drugdiscovery applications and animals used to model human diseases,including heart disease and atherosclerosis, that either naturallyexpress active CETP such as but not limited to, rabbits, hamsters,hedgehogs, ducks and certain fish or transgenic animals that aremodified to express CETP, such as mice.

[0034] Preferably, the bodily fluid sample is obtained from a subjectthat has been treated with a medication, medical protocol, dietaryregimen, or physical regimen that affects CETP activity. Non-limitingexamples of medications include drugs and naturally occurring compounds.The use of such samples permits the practice of the present invention todetermine the efficacy and/or effect of the treatment on CETP activityin the subject. In particularly preferred embodiments of the invention,the subject has been treated by administration of a CETP activityinhibitor, and said sample contains said CETP inhibitor, or said subjecthas been treated by administration of an inhibitor of CETP expression atthe transcriptional, translational, and/or secretion levels.

[0035] Particularly preferred in the practice of the invention is theuse of samples that have been frozen to disrupt endogenous lipoproteinsin the sample and reduce their effects on the assessment of CETPactivity in the samples.

[0036] The samples may also be from subjects having, or suspected ofhaving, a genetic abnormality associated with an abnormal level of CETPor CETP activity. Non-limiting examples include subjects that expressabnormal levels of CETP or a CETP inhibitor. The use of such samples incombination with the present invention permits the determination of thepresence of such a genetic abnormality.

[0037] In all embodiments of the invention, the level of CETP activityfound in a sample of bodily fluid may be compared to the activity foundin a sample from an untreated or normal subject, or a population ofsubjects, as reference. A diminution or enhancement of CETP mediatedtransfer in sample as compared to the reference identifies the sample ashaving an abnormal level of CETP activity which may require adjustmentby use of treatments that increase or decrease CETP activity as requiredby the extent and type of abnormality. Preferably, the untreated subjectis the same subject from whom the treated sample is obtained. The levelof CETP activity may also be compared to a standard to determine theamount of activity based upon the amount of substrate transferred byCETP.

[0038] The invention also provides for the ability to more accuratelyassess CETP activity despite the presence of endogenous modulators(inhibitors or activators) of CETP activity. Such endogenous modulatorsmay be naturally present and/or induced or suppressed by the treatmentof a subject as described above. The endogenous modulators may also actat the level of affecting CETP expression. The effect of modulators onCETP activity may not be reflected in determinations of CETP activity ina bodily fluid sample if the sample is diluted because the modulator maybe diluted beyond its ability to affect CETP activity as present in thebodily fluid. Therefore, the invention provides for the determination ofCETP activity without significant dilution of a bodily fluid sample.

[0039] By significant dilution of a bodily sample, it is meant dilutionof the sample by more than an equal volume of another solution. Stateddifferently, dilution of a sample beyond 50% v/v is significant withrespect to the present invention. Therefore, the present inventionprovides methods that determine CETP activity in solutions, or reactionmixtures, comprising at least 50% v/v of a bodily fluid sample. Morepreferably, the invention is practiced with solutions comprising atleast about 60%, at least about 70%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99% v/vof a bodily fluid sample. Of course the limitation on the maximum ratioof sample to total reaction mixture volume is the volume of the othercomponents in the reaction volume. The other components are simply thedonor and acceptor reagents as described herein, which may be formulatedto be in small volumes that do not significantly dilute a bodily fluidsample upon addition thereto.

[0040] The reaction mixtures of the invention permits CETP activity tobe detected. CETP ligands include two neutrally charged or non-polarlipids, namely, cholesteryl esters (“CE”), triacylglycerol, andtriglycerides (“TG”). These hydrophobic, neutral lipids are presentwithin the core of lipoprotein particles, including, but are not limitedto, high density lipoproteins (“HDL”), low density lipoproteins (“LDL”)and very low density lipoproteins (“VLDL”). Most lipoproteins are freelycirculating in the plasma of an organism. CETP transfers the two neutrallipids CE and TG from one lipoprotein particle, the donor, to anotherlipoprotein molecule, the acceptor.

[0041] CETP is thus a highly specialized protein that possesses transferactivity. In some animal and human diseases, especially heritablegenetic disorders, there is a deficiency in or over production of CETPthat leads to a phenotypic disorder. Exemplary phenotypic disordersinclude extremely high levels of HDL cholesterol in animals and humansexhibiting CETP deficiency, hyperalphalipoproteinemia associated with agenetic deficiency of plasma CETP, obesity associated with high levelsof CETP activity, low levels of HDL in patients exhibiting high CETPactivity, null mutations of CETP associated with markedly increasedlevels of HDL-cholesterol, accelerated development of artheroshcleroticlesions in organisms expressing CETP genes, and hypertriglyceridemia andlow HDL-C lipoprotein phenotypes associated with transgenic expressionof CETP genes.

[0042] There are genetic disorders in which CETP is either totallyineffective or has an increased or decreased activity. In these geneticdisorders the CETP may be affected in primary, secondary, or tertiarystructure. The incorrect amino acid is the result of a gene mutation inthe DNA coding for the respective enzyme, an error in transcription ofthe respective enzyme, or translation of the CETP protein. Geneticalterations can make CETP more efficient or less efficient. Theresulting increase in efficiency of CETP may have a harmful effect onthe subject animal in that increased CETP activity may lead to theconditions referred to above, and in particular is a risk factor fordeveloping atherogenic lesions.

[0043] A non-radioactive method of simplifying the screening for defectsof the gene coding for CETP affecting the activity of CETP comprisesobtaining a source or sample of CETP containing bodily fluid from asubject, such as a mammal. In some embodiments of the invention, asource of CETP also includes recombinant organisms, and extractstherefrom, having a gene coding for CETP or variant thereof,incorporated into the organism's genome with expression thereof.Recombinant organisms include, by way of example, transgenic mice, andalso other transgenic mammals.

[0044] The method further includes incubating for an effective timeperiod the sample in a non-radioactive CETP assay to obtain an incubatedmixture. The incubation preferably permits CETP transfer activity totransfer non-radioactively labeled neutral lipid from a donor particleto an acceptor particle. The label is preferably fluorescent and selfquenching. The components used to produce the incubated mixture arepreferably formulated as reagents for kits comprising instructions forthe practice of the methods of the invention.

[0045] The method further includes measuring the CETP enzyme activity ofthe incubated mixture to determine a CETP activity value, and comparingthe mixture's CETP activity value to a predetermined standard value.

[0046] A non-radioactive method of determining the efficacy, in a bodilyfluid, of a compound that inhibits CETP activity is provided by thepresent invention. In the case of excessive activity of CETP, a compoundinhibitor may be administered to a subject to inhibit CETP activity. Theinhibition of CETP may occur at different levels, including, but notlimited to direct inhibition of CETP activity or the inhibition of CETPexpression.

[0047] Two types of CETP activity inhibitors are irreversible andreversible inhibitors. Irreversible inhibitors combine with and/ordestroy the functional group of the CETP enzyme that is necessary forthe enzymes' transfer activity and dissociate very slowly from the CETPenzyme. Further, there are two types of reversible inhibitors of CETP,namely, competitive and non-competitive inhibitors of these enzymes.Competitive inhibitors compete with the substrate for binding to theactive site of CETP. However, once bound the competitive inhibitorcannot be transferred by CETP in the same way as a substrate can. Hence,competitive inhibition can be reversed by increasing substrateconcentration. Generally, competitive inhibitors of CETP resemble thenormal substrate in three-dimensional configuration. As a result of thissimilarity the competitive inhibitor “fools” CETP into binding to it.The effects of both irreversible and reversible inhibitors are moreaccurately assessed by use of the present invention because the effectsof both are subject to dilution.

[0048] Organisms having increased CETP transfer activity are treatedwith inhibitors of CETP activity as discussed herein, changes in dietthat affect CETP activity, or a combination thereof. The efficacy ofthese treatments on CETP activity is then evaluated using kits andmethods as described herein. To accurately evaluate the effects of thesetreatments, the method used should not interfere with the concentrationof the treatment article under test. The present invention allows CETPactivity measurements to be made without significantly changing theconcentration of inhibitors in a bodily fluid sample.

[0049] The method and kit of the present invention also facilitates andsimplifies the determination of the efficacy of a compound that inhibitsthe transcription/translation of a gene coding for CETP. For example, acompound that inhibits transcription/translation of a gene coding forCETP includes a segment of DNA/RNA having a complementary sequence tothe gene segment coding for the respective enzyme. Complementarysequences used as inhibitors are constructed using standard geneticengineering techniques. By way of example, a segment of “antisense” RNAis constructed or expressed to be complementary to CETP mRNA. Theorganism is given a treatment comprising of the complementary segment ofRNA. The complementary segment of RNA base pairs with the CETP mRNA andtranslation of the CETP is modified. The effectiveness of suchinhibitors of CETP expression is also assessed by the present inventionbased upon the CETP activity before and after, or in the presence andabsence of, use of the inhibitor.

[0050] Constitutive enzymes are those present in organisms at more orless constant amounts, and induced enzymes vary in concentration.Induced enzymes are regulated in concentration in organisms by enzymeinduction and repression. Induced enzymes are present in an organism intrace amounts but increase many (even a thousand) fold when the enzyme'ssubstrate is detected by the organism. An agent capable of inducing thesynthesis of an enzyme is an inducing agent. CETP is an enzyme whosegene is subject to dietary regulation and diet induced changes in lipidmetabolism. Atherogenic diets, e.g. those high in cholesterol, arecapable of inducing the synthesis of CETP and increasing the riskassociated with the formation of artherosclerotic lesions.

[0051] A dietary modification regimen is prescribed by a healthcareprofessional including a doctor, nurse, dietician and the like thatdirectly affects the induction, activity, or combination thereof, ofCETP. The dietary modification regimen is used as a treatment todirectly or indirectly affect the level of CETP activity. Prior to thedietary modification regimen, a baseline CETP value is determined for apatient using the method and kit described herein. The baseline CETPvalue is compared to a pre-determined range of values considered normalfor the patient's phenotype, age, gender, and/or genotype. Where thepatient's CETP value is outside the norm, a treatment affecting CETPactivity is recommended. The treatment may be a dietary modificationregimen, administration of a compound that inhibits CETP, administrationof a compound that affects the transcription/translation of CETP, orcombinations of these treatments.

[0052] In the case of a dietary modification regimen, the patientfollows the regimen over a predetermined period of time. The treatmentaffects the induction of CETP. Where the treatment comprises anon-atherogenic diet regimen and/or physical exercise, CETP activity inbodily fluids are expected to decrease, and there is a decreased riskfactor for developing atherosclerosis. After or during treatment, asample of the patient's bodily fluid may be obtained and tested inaccordance with the present invention to assess CETP activity anddetermine a new value for the activity. Further treatment may beprescribed to further affect CETP activity as needed.

[0053] In contradistinction to CETP induction, there is CETP enzymerepression. Enzyme repression involves the “turning off” of thesynthesis of the CETP enzyme upon the addition of a compound or upontreatment of a patient with the compound. The CETP repression concept iscentral to the principle of cell economy in that once high levels ofCETP enzyme are no longer needed in an organism, the respective enzymesare no longer made.

[0054] CETP enzyme regulation is in the form of transcriptional control,translational control, and control of secretion or export into theextracellular environment. Transcriptional control relates to thecontrol of the initiation and/or rate of transcription of the genescoding for CETP into their corresponding mRNAs. Translational controlinvolves the control of the initiation and/or rate of synthesis of theCETP polypeptide chain from its respective mRNA template. There areregulatory genes coding for regulatory proteins of CETP called CETPrepressors. The CETP repressor binds to the DNA segment coding for CETPcalled the CETP operator. Further, a CETP inducer can bind to a CETPrepressor to release the CETP repressor from the DNA binding site. Themethods and kits described herein are used to measure the efficacy ofCETP repressors, CETP inducers, compounds affecting the transcription ofCETP, compounds affecting the translation of CETP, and compoundsaffecting the secretion or export of CETP.

[0055] As with CETP induction, the application of the assay methodduring a course of treating a subject via CETP repression can beevaluated by the ability of the treatment to control the levels and/oractivity of CETP in the subject. The treatment may be modified toincrease or decrease the level of repression based upon the level ofCETP activity as determined by use of the present invention.

[0056] Donor and Acceptor Particle

[0057] As used herein, a “donor” particle refers to the particleresponsible for donating cholesteryl ester or other neutral lipid toCETP for transfer. An “acceptor” particle refers to the particle in anactivity measurement system (assay) responsible for acceptingcholesteryl ester or other neutral lipid transferred by CETP. Stateddifferently, a cholesteryl ester or other neutral lipid substrate to betransferred by CETP is resident in a donor particle, while an acceptoris the destination to which the substrate is transferred.

[0058] Preferred substrates for use in the present invention arecholesteryl esters (“CE”) and triglycerides (“TG”). Other potentialsubstrates may be tested in the assay of the invention to ascertaintheir ability to be transferred by CETP. The substrates are preferablylabeled in a manner that permits the detection of their transfer from adonor to an acceptor. Preferred labels are those that are detectableupon transfer away from a donor particle, such as self-quenching labelsthat exhibit enhanced fluorescence once they have been transferred awayfrom a concentrated label environment, such as that of a donor particle.Such labels are particular preferred in the case of homogenous assayformats where donor and acceptor are not separated from each other.Non-limiting examples of such fluorescent labels include5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacen (BODIPY® fromMolecular Probes, Inc. of Eugene, Oreg.), fluorescein, dansyl,rhodamine, or N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino (NBD) which canbe readily coupled to CETP substrates.

[0059] If the assay is performed in a heterogeneous format, virtuallyany label can be used because the acceptor and donor particle willultimately be separated. For example, the acceptor itself might becoupled to a member of a specific binding pair such as an antibody orfragment thereof or biotin and removed from the reaction mixture atvarious times and the level of label assessed. Thus, in this instance,the substrate may be labeled with a radioisotope, a fluorescent moiety,an enzyme (which then can be assayed using standard enzyme-based assays)or any other suitable label known in the art.

[0060] The donor particle is preferably an emulsion obtained by use of asuitable phospholipid emulsifier. Non-limiting examples includephosphatidylcholine and phosphatide extract. The substrate to betransfer may be dispersed throughout the emulsion or be concentratedwithin a portion of the emulsion. The emulsions may, of course, compriseother components, but such components are preferably not substrates forCETP transfer activity. The ratio of the components to a substrate maybe found by routine optimization. In particularly preferred embodimentsof the invention, the donor emulsion is as disclosed herein and does notcontain apoA-I protein.

[0061] The acceptor is typically an emulsion comprising lipoproteinparticles. Non-limiting examples include Intralipid™ and acceptorsprepared from fresh human plasma. Other examples of donors and acceptorsare provided in U.S. Pat. Nos. 5,770,355; 5,618,683; 5,585,235 and6,174,693. The ratio of acceptor to donor is kept appropriately highsuch that intra-donor particle transfer is not appreciable.

[0062] The donor and acceptor emulsions may be maintained in bufferedsolutions for a time prior to use in the detection of CETP activity. Thedetection of activity may be made after sufficient time has elapsedafter combination of donor, acceptor, and the CETP activity of a bodilyfluid sample to effect transfer, typically about 5 or 15 to about 90minutes, preferably about 45 to about 90 minutes, at about 37° C. Thetransfer can be monitored by the transfer of label as described above atvarious times after combination and mixing. The signal is monitored in amanner appropriate to the choice of label; in a preferred embodiment,the enhancement of fluorescence of a self-quenched label is measured ina homogeneous assay.

[0063] Method 1 to Prepare Donor

[0064] A self-quenching fluorescent neutral lipid, such as cholesteryllinoleate or other cholesteryl ester labeled withN-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino (NBD) to yield a moleculeNBD-CE, is emulsified by a suitable emulsifier such as phospholipid,like phosphatidylcholine (PC). Although the term emulsify is exemplifiedby a specific technique below, the present invention is concerned withincorporating the NBD lipid into an emulsified particle efficiently soas to achieve self-quenching emission characteristics of the label.There are many techniques known to emulsify hydrophobic or non-watersoluble compounds, such as, NBD-CE or NBD-triglyceride (NBD-TG), andmany compounds that will act as emulsifying agents.

[0065] Referring to FIG. 1, the emulsion 10 is prepared by sonicating 20micromoles of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino (NBD) labeledneutral lipid (CE or TG) 13, with 13 mg of phospholipid (PL) 11, at apower output just under that which causes the sonic probe to cavitatewithin the sample. A temperature above the melting point of the mixtureof component lipids is maintained for 45 minutes in a buffer of 10 ml,0.1M KCl/10 mM trizma-HCl, pH=8. The sonicated mixture is rapidly cooledto a temperature of 40 degrees Celsius. Ten (10) mg of apolipoproteinapoA-I 15 in 2.5 molar urea is added in less than 1 ml over 15 min. at asonication power half of that used for the high temperature sonication.

[0066] For applications of the present invention requiring a particlesimilar to HDL, the resulting emulsion is ultracentrifuged at a densityof 1.063 g/ml with 1.21 g/ml underlay and a 1.006 g/ml overlay. The HDLdensity class particle may be harvested from the 1.063 g/ml middle zone.This particle utilizes apolipoprotein apoA-I for stabilization, similarto physiological conditions. Stabilization of the fluorescent donorparticle may also be accomplished with synthetic, amino acid peptides orcasein.

[0067] Method 2 to Prepare Donor

[0068]FIG. 2 shows the particle 28 produced by a second method offluorescent neutral lipid donor synthesis. In this method, theemulsifier (PC) of Method 1 is replaced by phosphatide (PL) 29 extract.The phosphatide extract is derived from egg yolk or soybean, andincludes all phospholipids associated with each source, in particular:lecithin>60%, phosphatidyl-ethanolamine>15%, lysolecithin<4%. Theextract stabilizes the emulsion by providing charged emulsifyingphospholipids 29. The charged phospholipids emulsify the fluorescentcore comprised of NBD-CE 27, producing a net charge associated with eachparticle. The net charge associated with each particle causes repulsionbetween the fluorescent donor particles thereby preventing fusion of theparticles over time. The apolipoprotein apoA-I of the previous method ofdonor particle synthesis is eliminated by use of the PL extract. Thesonication is performed at 63 degrees centigrade (C.) to 68 degrees C.

[0069]FIG. 2 illustrates a synthetic or synthesized donor particlerepresentative of an emulsion. The NBD-labeled neutral NBD-CE 27contained within the core of the synthetic particle will not yieldsubstantial fluorescent emission intensity when illuminated withexcitation wavelength. Instead, the energy of the excited state isdissipated in radiationless energy transitions upon collision with otherNBD-CE molecules. The non-fluorescent loss of energy is dependent uponmolecular interactions associated with the core sequestered NBD-neutrallipid.

[0070] The monolayer of PL molecules 29 of FIG. 2, in the syntheticparticle is comprised of a polar head group and non-polar hydrophobictail. The conditions under which the emulsification process is performedenables the non-polar or hydrophobic tail of the PC molecule topartition with the hydrophobic NBD-neutral lipid, NBD-CE 27 of FIG. 2.The partitioning of hydrophobic constituents of the co-sonicationmixture traps the NBD-neutral lipid into a small area relative to thearea of the aqueous phase. The PC emulsified NBD-neutral lipidcomponents are in stable non-aqueous or hydrophobic environment at highconcentration with respect to collisional proximity and accordinglyyield little fluorescence intensity.

[0071] Method to Prepare Acceptor

[0072] Fresh human plasma has an initial density of 1.006 gm/ml. Apo-Bcontaining lipoproteins comprised of low density lipoprotein and verylow density lipoprotein are excellent acceptor substrates for CETP.Therefore, according to the invention, to fresh human plasma is addedsolid sodium bromide (NaBr) to adjust the density of the plasma from1.006 to 1.063 gm/ml (72.4 gms of NaBr to 940 mls of plasma). The plasmais ultracentrifuged at 1.063 gm/ml in a Beckman 50.2 rotor at 40,000 rpmfor 48 hours.

[0073] After ultracentrifugation, a turbid top layer, clear middle layerand turbid bottom layer will result. The top layer is the density lessthan 1.063 gm/ml layer while the turbid bottom layer is the densitygreater than 1.063 gm/ml layer. The top layer is removed and containsall the apolipoprotein B containing particles from the plasma. Theseparticles are relatively large spherical particles rich in neutrallipids such as triglycerides (TG) which provide an available substratefor CETP to hetero-exchange TG from acceptor for substrate (such asNBD-CE or BODIPY®-CE) from a donor. FIG. 3 illustrates the type ofparticles associated with the density less than 1.063 gm/ml top layer.The apo-B containing particle 32 includes apo proteins 31, neutrallipids 33, such as TG's and CE's and an outer layer comprised ofphospholipids 34. This fraction of particles is preferably used as theacceptor in the present invention and normalizes any differences inacceptor concentration in the plasma samples to be measured. A donorparticle without apoA-I is preferred because it will not compete withendogenous apoA-I containing lipoproteins which would alter the specificactivity of the donor.

[0074] While the above has been described with respect to human plasma,lipoprotein containing plasma from other animals may also be used toprepare acceptors for use in the present invention by routine methods.

[0075] Other Acceptors

[0076] In some embodiments, such as when fresh plasma is used by theinvention as a CETP containing sample, it may be preferable to utilizethe endogenous lipoproteins present in the plasma sample as acceptor.Therefore, a single bodily fluid sample from a subject can be used bothto provide the CETP activity to be assayed as well as the source ofacceptor for accepting transfer of substrate from a donor. Thisembodiment of the invention advantageously provides the ability toobviate the need to supply exogenous acceptor. The embodiment ispreferably practiced with donor comprising a labeled substrate thatpermits detection of CETP activity based upon transfer of substrate awayfrom the donor regardless of the nature of the acceptor.

[0077] Alternatively, a bodily fluid sample from a subject can be usedto provide one portion of the sample for processing as described aboveto prepare acceptor. Another portion of the sample can be used as theCETP containing sample to be tested. However, the presence of apo-Bcontaining lipoproteins in the CETP containing sample may introduceinaccuracies into the methods of the present invention because theselipoproteins may also act as acceptors. Therefore, the invention alsoprovides for the optional disruption of endogenous lipoprotein particlesin CETP containing samples. Such samples may be frozen, optionally at−40 or −80° C., to disrupt the integrity of endogenous lipoproteinparticles. Freezing lipoproteins will decrease their importance as asubstrate to CETP. Freezing of the samples will also allow long termstorage of the samples without a loss of CETP activity or decompositionof inhibitor(s) present in the sample.

[0078] Such frozen samples may be thawed and combined with the densityless than 1.063 gm/ml fraction of plasma as prepared above to normalizethe acceptor population among the samples. Further, donor particlewithout ApoA-I is used as the preferred substrate containing donor forassessing CETP transfer activity. CETP prefers a donor emulsion withoutapoA-I over endogenous HDL or frozen-then-thawed HDL.

[0079] Kits

[0080] The components useful in the invention can be provided in theform of a kit which contains at least a container which provides labeleddonor particles, preferably as an emulsion, and instructions or labelindications for its use in the methods of the invention. The kitoptionally further contains a container which contains an acceptor,preferably prepared from plasma as described above. If desired, only thecontainer of labeled donor may be provided, along with instructions forthe preparation of an acceptor emulsion or for the use of lipoproteinspresent in a CETP containing sample as acceptor.

[0081] Applications

[0082] The assay of the present invention may be used to assess amedical condition related to CETP activity. These conditions include butare not limited to heart disease and atherosclerosis,hypoalphalipoproteinemia, abnormal ratios of plasma apo A-I to apo B,increased secretion and levels of ApoB-containing lipoproteins, coronaryartery lesions, diabetes, obesity, and disorders involving cholesterylester deposits and transfer.

[0083] The invention may be used to screen CETP activity in plasma frommammals, including humans, that have been treated with an inhibitor ofCETP activity or measure the effects of an endogenous inhibitor of CETPactivity. The invention allows CETP activity to be determined on abodily fluid sample derived from a test subject without dilution of thesample which would change the concentration of inhibitor present in thesample. A variety of fluids may be used, depending on the interest ofthe skilled person and the presence of CETP activity. Exemplary CETPinhibitors include the following compounds: a)7[4′-trifluoromethyl-biphenyl-2-carbonyl)amino]-quinoline-3-carboxylicacid amides; b) Oxy substituted4-carboxyamino-2-methyl-1,2,3,4-tetrahydroquinolines; c)4-carboxyamino-2-substituted-1,2,3,4-tetrahydroquinoline; d) Substitutedbiphenyls; e) pyridines; and f) tetrahydroquinolines.

[0084] The following is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what is regarded as the invention nor are they intended torepresent that the experiments below are all or only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, temperature is in degrees Celsius, and pressure is at or nearatmospheric.

EXAMPLE 1

[0085] Determination of CETP Activity

[0086] A method for CETP activity measurement according to the presentinvention comprises: 1) mixing 7 μl of a donor particle emulsion with 30μl of isolated human plasma at density less than 1.063 gm/ml as theacceptor; 2) incubating 4 μl of the mixture of 1) with 50 μl of apreviously frozen-then-thawed plasma sample to be tested for CETPactivity for 45 to 90 minutes at 37 degrees Celsius; and 3) measuringthe fluorescent emission intensity of the mixture from 2). Thepercentage of plasma is about 93% v/v in the mixture.

[0087] Alternative exemplary amounts include donor emulsion containingapproximately 1.9×10⁻¹⁰ moles of NBD-labeled neutral lipid mixed with 10to 30 microliters of acceptor (density<1.063 gm/ml plasma fraction) andusing 4 microliters of this mixture to 50 microliters offrozen-then-thawed plasma sample to be tested and the fluorescence at538 nanometers (nm) is read at an excitation wavelength of 465 nm in astandard laboratory plate reading fluorimeter.

[0088] Referring to FIG. 5, the experiment was performed with 50 μlsamples of frozen-then-thawed human plasma that were pre-treated with aCETP inhibitory monoclonal antibody. The antibody pre-treatments weredilutions of stock mAb in 2 μl total Ab volume added to each 50 μlplasma sample. Following the pre-treatments, the plasma samples wereplaced in a microplate and 4 μl reagent according to the invention wasadded. The samples were incubated for 90 minutes at 37° C. The assayswere read at an excitation wavelength of 465 nm and emission wavelength535 nm. The total assay volume was 56 μl. The percentage of pre-treatedplasma is about 89% v/v in the mixture.

[0089] All references cited herein, including patents, patentapplications, and publications, are hereby incorporated by reference intheir entireties, whether previously specifically incorporated or not.

[0090] Having now fully described this invention, it will be appreciatedby those skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation. The invention also includes all of the steps, features,compositions and compounds referred to or indicated in thisspecification (unless specifically excluded) individually, collectively,and any and all combinations of any two or more of said steps orfeatures.

1. A method to determine cholesteryl ester transfer protein (CETP)activity in a bodily fluid of a subject, which method comprises:obtaining a sample of bodily fluid from said subject; contacting saidsample with a donor, comprising a labeled CETP substrate, and anacceptor, to which said substrate may be transferred by CETP activity,to form a reaction mixture solution, wherein said sample comprises atleast about 89% v/v of said reaction mixture solution; and detectingtransfer of said substrate from said donor to said acceptor to determinethe activity of CETP in said fluid.
 2. The method of claim 1 whereinsaid substrate is labeled with a self-quenching fluorescent molecule. 3.The method of claim 2 wherein said self-quenching fluorescent moleculeis N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino (NBD),5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, fluorescein, dansyl,or rhodamine.
 4. The method of claim 3 wherein said molecule is NBD. 5.The method of claim 3 wherein said substrate is a cholesteryl ester, atriacylglycerol, or a triglyceride.
 6. The method of claim 5 whereinsaid substrate is cholesteryl linoleate.
 7. The method of claim 1wherein said acceptor is an apo-B containing lipoprotein.
 8. The methodof claim 1 wherein said donor does not comprise apoA-I.
 9. The method ofclaim 1 wherein said sample has been frozen.
 10. The method of claim 1,further comprising computing, from a standard, the amount of substratetransferred.
 11. The method of claim 1, wherein said determining of theamount of label incorporated into the acceptor is performed on thereaction mixture without separating the acceptor from said reactionmixture.
 12. The method of claim 1 wherein said sample contains anendogenous CETP inhibitor or activator.
 13. The method of claim 12,further comprising comparing the level of CETP activity to apre-determined standard value indicating the presence of a risk factorfor heart disease, atherosclerosis, hypoalphalipoproteinemia, abnormalratios of plasma apo A-I to apo B, increased levels of ApoB-containinglipoproteins, coronary artery lesions, diabetes, and/or obesity.
 14. Themethod of claim 1, further comprising comparing the level of CETPactivity to a pre-determined standard value indicating the presence of agenetic defect in the gene encoding CETP that affects the amount oractivity of CETP protein in said sample.
 15. The method of claim 1wherein said subject has been treated by administration of a CETPactivity inhibitor, and said bodily fluid contains said CETP inhibitor,and/or has been treated with a non-atherogenic diet modification regimenthat affects physiological CETP activity.
 16. The method of claim 15wherein said inhibitor inhibits cholesterol synthesis and/or transfer,inhibits CETP expression, or is used to treat atherosclerosis.
 17. Themethod of claim 16 wherein said inhibition of CETP expression is byinhibiting the transcription or translation of CETP.
 18. A method todetermine the efficacy of a compound which modulates the activity ofCETP as present in a bodily fluid of a subject, which method comprisesobtaining a first sample of bodily fluid from said subject before, and asecond sample of bodily fluid from said subject after, administration ofsaid compound to said subject; contacting the first and second sampleswith a donor, comprising a labeled CETP substrate, and an acceptor, towhich said substrate may be transferred by CETP activity, to form firstand second reaction mixture solutions, respectively, wherein each samplecomprises at least about 89% v/v of said reaction mixture solutions; anddetecting transfer of said substrate from said donor to said acceptor todetermine the activity of CETP in each sample; wherein the efficacy ofsaid compound is determined by comparing the CETP activity of the secondsample to that of the first sample.
 19. A method to determinecholesteryl ester transfer protein (CETP) activity in a bodily fluid ofa subject, which method comprises: obtaining a sample of bodily fluidfrom said subject, wherein said fluid contains apo-B containinglipoproteins; contacting said sample with a donor, comprising a labeledCETP substrate which may be transferred to said lipoproteins by CETPactivity, to form a reaction mixture solution, wherein said samplecomprises at least about 89% v/v of said reaction mixture solution; anddetecting transfer of said substrate from said donor to saidlipoproteins to determine the activity of CETP in said fluid.
 20. A kitfor the determination of CETP activity in a bodily fluid sample, whichkit comprises a first container containing a suspension of donorparticles, which donor particles comprise labeled substrate andinstructions for performing the assay of claim 1, wherein said kitoptionally further comprises in a second container an acceptor fortransfer of the substrate.